Mesial Temporal Lobe Epilepsy: Pathogenesis,Induced Rodent Models and Lesions

Abstract

Mesial temporal lobe epilepsy (MTLE), the most common epilepsy in adults, is generally intractable and is suspected to be the result of recurrent excitation or inhibition circuitry. Recurrent excitation and the development of seizures have been associated with aberrant mossy fiber sprouting in the hippocampus. Of the animal models developed to investigate the pathogenesis of MTLE, post-status epilepticus models have received the greatest acceptance because they are characterized by a latency period, the development of spontaneous motor seizures, and a spectrum of lesions like those of MTLE. Among post-status epilepticus models, induction of systemic kainic acid or pilocarpine-induced epilepsy is less labor-intensive than electrical-stimulation models and these models mirror the clinicopathologic features of MTLE more closely than do kindling, tetanus toxin, hyperthermia, post-traumatic, and perinatal hypoxia/ischemia models. Unfortunately, spontaneous motor seizures do not develop in kindling or adult hyperthermia models and are not a consistent finding in tetanus toxin-induced or perinatal hypoxia/ischemia models. This review presents the mechanistic hypotheses for seizure induction, means of model induction, and associated pathology, especially as compared to MTLE patients. Animal models are valuable tools not only to study the pathogenesis of MTLE, but also to evaluate potential antiepileptogenic drugs.

Introduction

Nearly 2.5 million people in the United States of America suffer from temporal lobe epilepsy (TLE), a state of chronic neuronal hyperexcitability and hypersynchrony that is manifested as recurrent unprovoked partial seizures with or without secondary generalization (Cavazos, Jones et al. 2004). Mesial temporal lobe epilepsy (MTLE), one of the most common types of TLE, is characterized by seizure generation from the mesial temporal lobe (Falconer, Serafetinides et al. 1964; Margerison and Corsellis 1966).

In most affected patients, MTLE is thought to be initiated by lesions and functional alterations secondary to insults such as febrile convulsions, status epilepticus, encephalitis or trauma, which, after a 5 to 10-year latency period, generate spontaneous motor seizures (Engel 1993). MTLE can be subclassified histopathologically as paradoxical temporal lobe epilepsy (PTLE) or hippocampal sclerosis (HS) (Zaveri, Duckrow et al., 2001). No lesions are observed in PTLE, whereas in the 70% of MTLE patients with HS (aka mesial temporal sclerosis or Ammon’s horn sclerosis) lesions include neuronal degeneration, astrogliosis (Babb and Brown, 1986) and aberrant mossy fiber (MF) sprouting in the inner molecular layer of the dentate gyrus. The synaptic boutons of aberrant MF sprouts can be visualized histochemically with Timm’s stain (Danscher and Zimmer 1978), because of their higher zinc concentration, or by immunohistochemical labeling of the presynaptic growth-associated protein B-50 (aka GAP-43, neuromodulin, F1) (Proper, Oestreicher et al., 2000). Dynorphin immunohistochemistry (Schwob, Fuller et al., 1980; Lothman and Collins, 1981) facilitates identification and quantification of MFs because boutons contain large quantities of this opioid peptide. Biocytin and lectin tracer Phaseolus vulgaris leucoagglutinin (PHAL) are also useful for visualizing MFs (Sutula, Zhang et al., 1998; Wenzel, Woolley et al., 2000).

Because unilateral excision of the hippocampus effectively diminishes seizures (Ojemann, 1987; Spencer, 2002) and because lesions are found in the hippocampus of TLE patients (Babb and Brown, 1986), it is considered the seat of seizure generation. Therefore, most animal models are focused on reproducing and understanding the pathogenesis of HS.

TLE is mainly treated with antiseizure rather than antiepileptogenic drugs (symptoms, but not the disease, are treated). Nevertheless, 40% of TLE cases are refractory to medical treatment. Elucidation of the pathogenesis and course of TLE is needed to develop novel targeted therapies. A variety of rodent models have been developed to study the cellular and molecular mechanisms of MTLE. This review focuses on the leading mechanistic hypotheses and current rodent models of MTLE, with special reference to methods of induction and comparative neuropathological changes.

Hippocampal structures involved in epilepsy

Because a central role in epileptogenesis is attributed to the hippocampus, a brief review of anatomy, pathways and interaction of the hippocampus with other parts of the limbic system will facilitate understanding of the proposed mechanisms and pathologic changes of MTLE. An excellent review of hippocampal structures and pathways is provided by Goldensohn and Salazar (1986).

Important subanatomic locations affected in MTLE and its rodent models are included in Figure 1.

Briefly, the rodent hippocampus is a somewhat flattened, distinct, elongated structure between the mesial (medial) part of the temporal lobe and the underlying thalamus. In rats, the hippocampus extends caudally and ventrally from the dorsal midline just above the thalamus at Bregma -1.72 to the ventral subiculum at Bregma -6.84. Numerous fiber tracts connect the hippocampus with other parts of limbic system, via the circuit of Papez, as outlined in Figure 2. The hippocampus consists of two interlocking C-shaped areas called Ammon’s horn and the dentate gyrus, which are composed principally of pyramidal and granule cell neurons, respectively. Ammon’s horn has been divided into four sub-fields, CA1 through CA4. CA1 extends from the dorso-medial subiculum to a slightly thickened band, CA2. CA3 comprises the ventro-medial portion of the hippocampus. The terminal portion of CA3, as it courses into the dentate hilus, is sometimes referred to as CA4, although many authors simply refer to this terminal portion as CA3. CA3 neurons receive MF input from the dentate gyrus.

MFs are the axons of dentate granule cells, which normally synapse on the dendrites of CA3 pyramidal neurons. Dentate granule cells are oriented with their dendrites projected towards the molecular layer. A GABAergic population of neurons called dentate basket cells is intermingled with the dentate granule cells. Enclosed within the curvature of the dentate gyrus is the dentate hilus, which contains polymorphic neurons of CA4 extending from CA3 as well as the glutamatergic hilar mossy cells. Hippocampal pathways are outlined in Figure 1D. Several fiber tracts that connect the hippocampus with other parts of the limbic system, via the circuit of Papez are outlined in Figure 2.

Classification of seizures

This section reviews the types of seizure to facilitate understanding of the terminology used in subsequent sections. Epileptic seizures have been classified into two broad categories - partial (focal) and generalized, based on clinical signs. Partial seizures originate from a focus of neurons in one cerebral hemisphere and are subclassified as simple or complex. Simple partial seizures may be associated with sensory, somatosensory, autonomic, or psychic symptoms, but do not cause loss of consciousness and do not last more than one minute. In contrast, complex partial seizures are always associated with loss of consciousness and may last a few minutes. Both simple and complex partial seizures may gradually develop into secondary generalized seizures of no more than a few minutes’ duration. Primary generalized seizures result from paroxysmal discharges arising in both cerebral hemispheres that may manifest as tonic-clonic seizures with loss of consciousness (grand mal seizures) or generalized tonic or clonic seizures with or without loss of consciousness. Status epilepticus (SE), which can be either partial or generalized, is a state of continuous seizure activity for five minutes or longer. In this review, any prolonged seizure activity resulting from chemical or electrical injury or other stimuli will be classified as SE.

Proposed mechanisms of MTLE

In general, proposed mechanisms of MTLE are based on the unique anatomic relationship of the hippocampus to the limbic system and on receptor-based electrophysiological changes related to either recurrent inhibition or excitation.

The position of the hippocampus relative to other parts of the limbic system and its cross-talk with those parts may have a central role in seizure generation. The presence or absence of various surface receptors on neurons may impart resistance or susceptibility to selected hippocampal areas leading to neuronal degeneration and seizure generation.

As outlined in Figure 2, hippocampal circuitry is a part of a larger circuit known as the Papez circuit, which ultimately constitutes a part of the limbic system. It has been proposed that increased hippocampal activity goes out to other parts of the limbic system and returns as amplified input to the hippocampus via the Papez circuit, which can lead to generalized epileptic seizures. Although imaging studies have revealed abnormalities along the afferent and efferent pathways of the Papez circuit (Bronen et al., 1991; Oikawa et al., 2001), concrete evidence of a cause and effect relationship between the Papez circuit and generation of epileptic seizures is not available. In contrast, the role of excitatory and inhibitory receptors and/or their subunits is supported by various studies, as discussed next.

Shifts in the distribution of receptors on hippocampal neurons may also lead to increased hyperexcitability and/or neurodegeneration of selected neuronal populations. The excitatory neurotransmitter glutamate is released from presynaptic neurons and acts upon the ionotrophic glutamate receptors Kainate, AMPA and NMDA. Kainate and AMPA receptors cause fast depolarization of neurons by allowing influx of monovalent cations such as Na⁺ and K⁺, whereas NMDA receptors effect a slower depolarization by allowing influx of divalent cations, primarily Ca⁺⁺. Ionotropic glutamate receptors are composed of multiple combinations of subunits.

While there is little information to support the role of NMDA receptors or their subunits in causing increased hippocampal hyperexcitability or neurodegeneration, data do suggest that the absence or decreased expression of the GluR2 subunit in AMPA receptors may play a role in neuronal hyperexcitability and/or neurodegeneration. The presence of the GluR2 subunit in AMPA receptors controls the influx of Ca⁺⁺into the neurons. GluR2 knockdown caused by administration of antisense oligonucleotide probes in the dorsal hippocampus of adult rats caused degeneration of CA3 neurons (Friedman et al., 2003). Decreased expression of GluR2 subunits on CA3 neurons in a kainic acid (KA)-induced rat model of MTLE implicates their absence in CA3 neuronal degeneration (Sommer et al., 2001).

Similarly, decreased GluR2 expression by hippocampal CA1 neurons may lead to neuronal hyperexcitability in a hypoxia-induced perinatal rat model of epileptogenesis (Sanchez et al., 2001). Increased expression of kainate receptor subunit GluR6 by administration of HSV-1 vector bearing GluR6 into rat hippocampus caused spontaneous seizures, hyperexcitability in CA1 cells and loss of CA1, hilar, and CA3 neurons (During et al., 1993). The inhibitory receptors, GABA and adenosine A1 (A1Rs), are also implicated in some forms of seizure generation.

The principal inhibitory neurotransmitter in the brain, gamma amino butyric acid (GABA), acts on two types of receptors, namely GABA_A, the ligand-operated ion channels, and GABA_B, the G-protein-coupled metabotropic receptors. GABA_A receptors are responsible for fast inhibitory post-synaptic potential (IPSP), whereas GABA_B receptors cause slower IPSP. Altered expression of GABA _A receptor subunits has been reported in both MTLE models (Poulter, Brown et al., 1999; Peng, Huang et al., 2004) and human patients (Loup, Wieser et al., 2000). Altered GABA_A receptor subunits probably decrease inhibition resulting in neuronal hyperexcitability.

Presynaptic GABA_B receptors suppress neurotransmitter release and have anticonvulsant or proconvulsant activities depending on whether they are located on glutamatergic or GABAergic neurons. Although binding of GABA_B receptor antagonists to CA3 pyramidal cells made the depolarizing GABA response excitatory and proconvulsive (Kantrowitz, Francis et al., 2005), the exact role of these receptors in seizure generation is unclear.

In addition to GABA receptors, A1Rs also have an inhibitory effect on the hippocampus. A1Rs inhibit adenylate cyclase activity (Dunwiddie and Fredholm, 1989) leading to increased K⁺ conductance (Greene and Haas, 1985) and decreased Ca⁺⁺ influx (Wu and Saggau 1994). The presence of numerous receptors on CA2 neurons as compared to CA1 or CA3 neurons (Ochiishi et al., 1999) probably make CA2 neurons resistant to glutamate excitotoxicity (Mattson and Kater, 1989) and status epilepticus (Young and Dragunow, 1995).

Regardless of their origin, the partial-onset seizures that are typical of MTLE are recorded as focal interictal epileptiform spikes or sharp waves originating at or near the seizure focus. These spikes are caused by a paroxysmal depolarization shift (PDS) in neurons. During PDS, prolonged calcium-dependent conductance causes depolarization of the neuronal membrane, resulting in threshold voltage and activation of voltage-gated sodium channels. Influx of sodium reduces the resting potential and causes firing of multiple sodium-dependent action potentials, followed by hyperpolarization with active transport of sodium ions out of the neurons. Synchronous PDS discharges fired by millions of neurons create an electrophysiologically detectable focal interictal epileptiform spike that manifests as partial-onset seizures (Babb, Kupfer et al., 1991; Franck, Pokorny et al., 1995; Masukawa, O’Connor et al., 1995; Zhang and Houser, 1999).

The literature contains many hypotheses of MTLE. This section will focus on the recurrent excitation and inhibition hypotheses considered most tenable by the authors, based on review of the literature and their own research. Both hypotheses focus on the dentate gyrus as the nidus for epileptogenesis.

Recurrent excitation hypothesis

One of the most widely accepted explanations of epileptogenesis, the recurrent excitation hypothesis, states that spontaneous motor seizures in MTLE are a consequence of hyperexcitability of dentate granule cells, resulting from alterations of circuitry due to aberrant MF sprouting. These recurrent loops probably induce dentate granule cells to stimulate each other, leading to generation of spontaneous motor seizures from the hippocampus (Figure 3). Studies of aberrant MF sprouting have been reviewed in various animal models of MTLE, such as KA-, pilocarpine-, and kindling-induced models (White, 2002). Under normal circumstances, mossy fibers from dentate granule cells traverse the dentate hilus and stratum lucidum of Ammon’s horn and form synapses with hilar mossy cells and CA3 pyramidal neurons. In patients with MTLE, aberrant MF sprouts have been found in the granule cell layer and the inner molecular layer of the dentate gyrus (Okazaki, Molnar et al., 1999; Buckmaster, Zhang et al., 2002).

Similar MF sprouts were detected in animal models of MTLE (Buckmaster, Zhang et al., 2002). These aberrant MF sprouts formed synapses with dendrites of granule cells leading to recurrent excitatory circuitry (Lothman, Stringer et al., 1992). In comparison to 61% of granule cells from rats exhibiting aberrant MF sprouting following pilocarpine-induced status epilepticus, only 13–16% of granule cells in control rats exhibited excitatory postsynaptic current (EPSC). This implicated MF sprouts in seizure generation (Obenaus, Esclapez et al., 1993; Houser and Esclapez, 1996). Histopathologic and ultrastructural findings in a pilocarpine-induced status epilepticus model showed that 93–96% of synapses of aberrant mossy fibers were formed with GABA-negative granule cells and fewer than 1 of 20 new synapses were formed with GABAergic interneurons. The resulting circuits were predominantly excitatory (Ribak, 1985; Robert, 1994; Sloviter, 1996; Menks and Sankar, 2002). Because dentate granule cells seldom have spontaneous epileptiform discharges, they form a barrier to the spread of seizures to other parts of the brain (Menks and Sankar, 2002); however, it is proposed that this recurrent excitatory circuitry overcomes this barrier.

Recurrent inhibition hypothesis

Results of animal models and human studies also support the hypothesis that dentate granule cells form a nidus for epileptogenesis secondary to loss of inhibitory neurons (Menks and Sankar, 2002), such as hilar mossy cells or dentate basket cells. Mossy cells are glutamatergic neurons in the dentate hilus, whereas dentate basket cells are GABAergic neurons of the dentate gyrus. The perforant pathway from the entorhinal cortex (EC) provides stimulatory input to dentate granule cells as well as to hilar mossy cells and dentate basket cells. Stimulation of hilar mossy cells and dentate basket cells causes feed-forward inhibition of the granule cells (Ratzliff, Howard et al., 2004). Glutamatergic mossy cells are stimulatory, whereas the glutamic acid decarboxylases (GAD)-positive inhibitory GABAergic neurons, also known as basket cells, are inhibitory, especially to dentate granule cells. In addition to innervating dendrites of the CA3 pyramidal cells, MF collaterals also innervate the hilar mossy cells.

Hence, stimulation of granule cells via the perforant path causes stimulation of hilar mossy cells, which in turn stimulate the GABAergic basket cells leading to inhibition of dentate granule cells, a phenomenon also termed feedback inhibition (Obenaus, Esclapez et al., 1993). Ablation of hilar basket cells caused dentate granule cell hyperexcitability in a trauma-induced rat model of TLE (Robert, 1994; Sloviter, 1996; Zappone and Sloviter, 2004). In situ hybridization detection of GAD mRNA revealed a marked reduction in the number of GAD-positive hilar neurons in a rat model of TLE (Buckmaster, Wenzel et al., 1996). However, this study could not detect GABAergic neurons in this cell population and indicated that basket cells may be resistant to epileptogenesis.

The “dormant basket cell hypothesis” is based on a loss of hilar mossy cells. In the normal hippocampus, hilar mossy cells induce inhibition via the basket cells. Loss of hilar mossy cells causes dormancy in inhibitory basket cells and loss of inhibition of the dentate granule cells (Figure 3) (Ratzliff, Howard et al., 2004). In contrast, others (Houser and Esclapez, 1996; Schwarzer, Tsunashima et al., 1997; Brooks-Kayal, Shumate et al., 1998) have found that mossy cells innervate granule cells rather than inhibitory basket cells and some investigators (Esclapez and Houser, 1999; Smith and Dudek 2001; Bausch and McNamara, 2004; Perez-Mendes, Cinini et al., 2005) suggested that loss of mossy cells causes decreased excitability of granule cells following perforant path stimulation. Because of these discrepancies, the recurrent excitation hypothesis is favored over the dormant basket cell hypothesis.

Background, Dosing Information, and Pathological Alterations in Rodent Models of MTLE

Many rodent models have been developed to study the pathogenesis of MTLE (Coulter, McIntyre et al., 2002; White, 2002; Buckmaster, 2004). Examples include chemoconvulsant and electrical stimulation-induced post-status epilepticus, kindling, tetanus toxin, hyperthermia, post-traumatic epilepsy, and perinatal hypoxia/ischemia (HI) models. These models have certain similarities to, and dissimilarities from, MTLE that impact their utility or relevance. Our literature search failed to find reports of genetic models of MTLE. Information on genetic models of other types of epilepsies (Consroe and Edmonds, 1979; Loscher, 1984; Lason, 1998) are available to readers interested in non-MTLE models of epilepsy. Selected protocols and hippocampal lesions in various models of MTLE are listed in Table 1.

Post-Status Epilepticus Models

Post-status epilepticus models closely mimic the clinical manifestations of MTLE in humans, in which an acute triggering process is frequently followed by a latency period with subsequent development of spontaneous motor seizures. An acute episode of seizures, also known as status epilepticus, is the triggering process in these animal models. The models are created by systemic or local administration of chemoconvulsants or electrical stimulation. In status epilepticus models, seizures are induced in rats or mice for 1 to 2 hours before status epilepticus is interrupted by an anticonvulsant. Rehydration or supplemental nutrition may be required to restore the rodents to a healthy state. After a few weeks, many will start exhibiting spontaneous, secondary generalized seizures.

Chemoconvulsants

KA (Tauck and Nadler, 1985; Okazaki, Molnar et al., 1999) and pilocarpine (Turski, Cavalheiro et al., 1984; Okazaki, Molnar et al., 1999) are the two of the most commonly used chemoconvulsants used to create status epilepticus models of MTLE. Hippocampal lesions in these models are similar to the HS observed in humans with MTLE (Turski, Cavalheiro et al., 1984; Ben-Ari, 1985; Cavalheiro, Leite et al., 1991; Sloviter 1996). In addition to loss of specific cell populations in Ammon’s horn, hippocampal alterations in these animal models and human patients include aberrant MF sprouting (Turski, Cavalheiro et al., 1984; Cronin and Dudek, 1988; Sutula, Cascino et al., 1989; Babb, Kupfer et al., 1991; Mello, Cavalheiro et al., 1993; Obenaus, Esclapez et al., 1993; Okazaki, Evenson et al., 1995) and other circuit rearrangements (Okazaki, Molnar et al., 1999).

Kainic acid

KA was among the first chemoconvulsants used to create rodent models of MTLE. KA [2-carboxy-4 (1-methylethenyl)-3-pirrolidiacetic acid]. It is a cyclic analog of L-glutamate and an agonist of ionotropic, non-NMDA glutamate AMPA and KA receptors. KA, initially isolated from seaweed in Japan and used as an ascaricide, is almost one hundred times more potent than L-glutamate at these 2 receptors. KA is commonly administered systemically or intracerebrally to cause sustained neuronal depolarization. This leads to seizure generation characterized clinically by changes in physical activity, stereotypic grooming, “wet dog” shakes, variably intense continuous tremors, and continuous clonus. Approximately 3 decades ago, KA was first used in a rat model of MTLE (Nadler, Perry et al., 1978; Ben-Ari 1985). Early studies used KA at a single dose from 12 to 18 mg/kg via the i.p., s.c., or i.v. route. The high mortality rate associated with these doses was significantly reduced and seizures were produced in more rats when treatment was modified from a single dose to multiple 5 mg/kg doses (Hellier, Patrylo et al., 1998). When our laboratory used a single dose of KA at 9 mg/kg in 7- to 8-week-old Fischer-344 rats, 95% of the rats survived; approximately 93% of survivors exhibited status epilepticus; and 80% developed spontaneous motor seizures (unpublished data). To reduce mortality, status epilepticus was halted after 1.75 hr by i.p. diazepam at 10 mg/kg and subcutaneous fluids were administered to dehydrated rats unable to drink water.

Disrupting seizure activity too early may reduce the intensity or delay the onset of recurrent spontaneous motor seizures and could affect the development of lesions (Sutula, Cascino et al., 1989; Brandt, Potschka et al., 2003; Pitkanen, Kharatishvili et al., 2005). In alternate protocols, KA was injected directly into either the hippocampus (Longo and Mello, 1998; Bragin, Engel et al., 1999; Okazaki, Molnar et al., 1999) or lateral ventricle (Nadler, Perry et al., 1978; Okazaki, Molnar et al., 1999). A lower mortality rate in comparison to systemically administered KA models is an advantage of administering KA directly into the brain. However, stereotactic administration of KA requires surgery, which is labor-intensive and time-consuming.

Many neuropathological features of the KA-induced rat (Suzuki, Makiura et al., 1997; Kralic, Ledergerber et al., 2005) and mouse (Nadler, Perry et al., 1978; Benkovic, O’Callaghan et al., 2006) models of MTLE mimic the changes observed in human MTLE (Lothman and Collins, 1981; Sloviter, 1996). KA caused necrosis and loss of neurons in hippocampal subfields CA1 and CA3 (Tauck and Nadler, 1985; Okazaki, Evenson et al., 1995; Chakravarty, Babb et al., 1997) and MF sprouting within the inner molecular layer of the dentate gyrus (Represa, Le Gall La Salle et al., 1989; Represa, Robain et al., 1989; Patrylo, Schweitzer et al., 1999; Proper, Oestreicher et al., 2000; Sommer, Roth et al., 2001). Figure 4 shows CA1 neuronal degeneration and subsequent aberrant MF sprouting in a MTLE model produced by a single subcutaneous dose of KA at 9 mg/kg in 7 to 8-week-old Fischer-344 rats. Neuronal necrosis was also detected in the piriform and olfactory cortices, amygdaloid nuclei, lateral septum, several thalamic nuclei (Ben-Ari, Tremblay et al., 1979) and neocortical layers III and VI (Pollard, Charriaut-Marlangue et al. 1994).

Several investigators (de Lanerolle, Kim et al., 1989; Sutula, Cascino et al., 1989; Houser, Miyashiro et al., 1990; Cohen-Gadol, Pan et al., 2004) administered KA i.p. and detected apoptosis by TUNEL (in-situ end-labeling) in the CA1, CA3, and dentate hilar neurons from 24 hrs to 4 weeks post KA administration. Hippocampal astrogliosis and microgliosis were confirmed by GFAP and MHC I and II immunohistochemistry, respectively. Neurogenesis, characterized by BrdU positivity, occurred in dentate granule cells between days 3 and 10 with a peak at day 5. Prominent features, such as neuronal loss and necrosis in the hippocampus and MF sprouting, were similar to those described in surgical specimens from MTLE patients (Turski, Cavalheiro et al., 1984). Balchen, Berg et al., (1993) reported subtotal-to-total pyramidal neuronal loss from both CA1 and CA3 following i.p. administration of KA at 10 mg/kg. Interestingly, if the dose of KA was increased to 20 mg/kg, severe damage was present in the CA3, but minimal to no damage in CA1 pyramidal neurons. These findings suggested that the increased dose of KA damaged the CA3 pyramidal neurons before excitatory input through the Schaffer collateral pathway could damage the CA1 pyramidal neurons.

The neuropathologic alterations produced by KA, although similar to those produced by pilocarpine (discussed below), overlap to an even greater extent with those reported in the surgically resected hippocampi of human patients (Sutula, Cascino et al., 1989; Babb, Kupfer et al., 1991). Furthermore, the electroencephalographic and behavioral alterations caused by KA (Lothman and Collins, 1981) were analogous to those observed in human patients with MTLE (Krumholz, Sung et al., 1995; Wakai, Ito et al., 1995) and were almost identical to those caused by pilocarpine except for the fact that pilocarpine produces more severe lesions in the neocortex and less severe lesions in the hippocampus (Turski, Cavalheiro et al., 1984). Therefore, KA is considered one of the best animal models of MTLE. High mortality is a disadvantage of some KA models, but can be minimized by lower doses, appropriately timed anticonvulsants and supportive therapy.

Pilocarpine

Similar to KA-induced models, pilocarpine treatment is frequently used to model MTLE in rats and mice. Pilocarpine is a muscarinic acetylcholine receptor agonist. Like KA, pilocarpine, especially in combination with lithium, reproduces many clinical and morphological aspects of MTLE in rodents; however, lesions are more prominent in the neocortex than in the hippocampus.

Pilocarpine was first used in rats (Turski, Cavalheiro et al., 1984) and mice (Fujikawa, 2003) to produce limbic seizures as observed in MTLE. Pilocarpine doses of 340 to 380 mg/kg usually induced status epilepticus within 20 to 30 minutes in adult rats (Garcia-Cairasco, Rossetti et al., 2004; da Silva, Regondi et al., 2005). Atropine was generally administered prior to pilocarpine treatment to negate any cholinergic effects. When lithium was administered subcutaneously at 3 mEq/kg prior to pilocarpine administration, it not only reduced the required pilocarpine dose to 30 mg/kg, but also produced epilepsy more reliably and was associated with a lower mortality rate (Clifford, Olney et al., 1987; Kubova and Moshe, 1994; Voutsinos-Porche, Koning et al. 2004).

Alternatively, pilocarpine can be injected directly into either the piriform cortex (Milhaud, Rondouin et al., 2003) or hippocampus (Furtado Mde, Braga et al., 2002). Status epilepticus in these models was blocked by anticonvulsants such as diazepam, usually administered within two hours after induction. As with the KA model, premature inhibition of status epilepticus may either reduce the intensity or delay the onset of recurrent spontaneous motor seizures (Lemos and Cavalheiro 1995; Fujikawa 1996; Gu, Lynch et al., 2004).

Neuropathological findings in the pilocarpine model included neuronal necrosis in the hippocampus, cerebral cortex and olfactory cortices, amygdala, thalamus, and substantia nigra. In rats, the number of necrotic neurons increased with the duration of status epilepticus (Clifford, Olney et al., 1987). Neuronal necrosis occurred in the hippocampal subfields dorsal CA2 and ventral CA1 and CA3, dentate hilus, as well as in amygdaloid nuclei, layers 2–4 of piriform and entorhinal cortices, layers 2–5 of frontoparietotemporal cortex, lateral septal nuclei, thalamic reuniens nucleus, and caudate putamen. The number of necrotic neurons appeared to increase up to 24 hrs post-administration, but decreased at 72 hrs, perhaps as a result of loss (drop-out) of dead neurons. Similar lesions were observed when the high dose of pilocarpine was replaced with low doses of lithium and pilocarpine (Mello, Cavalheiro et al., 1993). With either protocol, additional neuropathological changes included dispersion of the granule cell layer of the dentate gyrus and Timm’s stain-positive supra- and intra-granular MF sprouting as early as 4 and 9 days, respectively.

These changes reached a plateau by 100 days (White 2002). In a similar study in rats, extensive, immediate neuronal damage occurred within the substantia nigra reticulata, CA1 field of the hippocampus, the piriform cortex, and the reuniens and paratenial nuclei of the thalamus and persisted for up to 50 days. These progressive alterations resembled those in limbic status epilepticus evoked by electrical stimulation of the olfactory cortex or basal amygdaloid nucleus (Borges, Gearing et al., 2003) and by KA, as discussed above.

In a CF1 mouse pilocarpine model, neuronal degeneration was evaluated between days 1 and 31 post status epilepticus. Extensive loss of hilar neurons in the hippocampus occurred within 6 hrs, and was followed by varying degrees of pyramidal cell damage in CA1 and CA3 subfields from days 1 to 31, and neuronal loss in the thalamus, striatum, piriform cortex, and amygdala from days 3 to 31. Increased numbers of β2 microglobulin (an MHC class I molecule)-positive microglial cells and GFAP-positive astrocytes appeared in all damaged areas after 3 and 10 days post status epilepticus, respectively (Borges, Gearing et al., 2003). Robust MF sprouting was present in the inner molecular layer at 4 to 8 weeks following pilocarpine administration in CD-1 and C57BL/6 mice (Turski, Cavalheiro et al., 1984; Obenaus, Esclapez et al., 1993; Cavalheiro, Santos et al., 1996; Shibley and Smith 2002). Neuronal loss and neurogenesis have also been described in other studies in rats and mice (Fifkova and Van Harreveld, 1977; Vicedomini and Nadler, 1987; Sloviter, 1996; Gorter, van Vliet et al., 2001; Brandt, Ebert et al., 2004).

The electroencephalographic and behavioral alterations caused by pilocarpine (Turski, Cavalheiro et al., 1984) are analogous to those caused by KA (Lothman and Collins 1981) and in human MTLE (Krumholz, Sung et al., 1995; Wakai, Ito et al., 1995). However, as noted under the KA model discussion, the distribution of lesions in the KA models more nearly resembles human MTLE than does the pilocarpine models. As in KA models, high mortality is a disadvantage of pilocarpine models. However, mortality can be limited by decreased dosage and appropriately timed anticonvulsant and supportive therapy.

Electrical Stimulation

Compared to KA and pilocarpine, electrical stimulation is used less often as a model of MTLE. However, this model produces clinical signs and lesions comparable to those of the KA and pilocarpine models. Electrical pulse trains have been used to stimulate different regions of the brain to produce self-sustaining seizures without prior kindling or administration of chemoconvulsants. Repetitive tetanic stimulation of hippocampal afferents such as the perforant path (Schwob, Fuller et al., 1980; Vicedomini and Nadler 1987; Sloviter 1996; Ribak, Tran et al., 2000; Gorter, van Vliet et al., 2001; Mazarati, Lu et al., 2004), hippocampus (Racine 1972; Lothman and Collins 1981; Vezzani, Conti et al., 1999; Bastlund, Jennum et al., 2005), or amygdala (Racine 1972; Cavazos and Sutula 1990; Tilelli, Del Vecchio et al., 2005) has been used to produce status epilepticus. Of the various protocols for electrical stimulation, the method reported by Vicedomini (1987) is the prototype. Unanesthetized rats were stimulated with 0.2 to 0.4 millisecond monophasic rectangular pulses at 20Hz with 10 seconds train duration and 30 seconds inter-train interval through chronically implanted electrodes in angular bundles or fimbria. Electrical stimulation was stopped when 10 consecutive trains each produced 30 seconds of hippocampal after-discharge. Rats were then monitored for self-sustained electroencephalographic seizure activity; 85% exhibited status epilepticus within 7 hrs.

Sloviter (1996) described the neuropathologic changes following electrical stimulation of the brain. Unilateral sustained electrical stimulation of the perforant path in anesthetized rats caused necrosis of CA1 and CA3 pyramidal cells and hilar neurons, whereas CA2 neurons were generally unaffected. Based on unilaterally or bilaterally evoked granule cell discharges, the neuronal damage was likewise unilateral or bilateral (Tilelli, Del Vecchio et al., 2005). In addition to the hippocampal lesions, electrical stimulation of the amygdala caused neuronal necrosis in the piriform cortex (Gorter, van Vliet et al., 2001). Electrical stimulation of the angular bundle caused extensive aberrant MF sprouting in the inner molecular layer of the dentate gyrus (Brandt, Glien et al., 2003). Self-sustained status epilepticus induced by high intensity (700 μA) pulsed-train electrical stimulation of the basolateral nucleus of the amygdala produced neuronal necrosis in the ipsilateral amygdala, piriform cortex, entorhinal cortex, endopiriform nucleus, and mediodorsal thalamus in rats. Aberrant mossy fiber sprouting was also present in the inner molecular layer of the dentate gyrus (McIntyre, Nathanson et al. 1982). Depending upon the area of the brain and the intensity of pulse train electrical stimuli, these models show minor variations in the site and the severity of brain lesions. However, all protocols were generally successful in producing the hippocampal lesions of MTLE.

Histopathologic findings are comparable to those in KA and pilocarpine-induced models and MTLE patients. However, electrode implantation is cumbersome and labor-intensive, which reduces the desirability of this model relative to chemoconvulsant models.

Kindling Models

Kindling triggers epileptic seizures by repeated small electrical or chemical stimulation to the brain. Kindling models differ from post-status epilepticus models, as repeated subconvulsant levels of either electrical stimulation or chemoconvulsants do not produce SE immediately following the treatments. Moreover, once the animal is fully kindled, “evoked” seizures can be incited, rather than the spontaneous motor seizures observed in MTLE patients and post-status epilepticus models. The resulting lesions, therefore, differ from those in the post-status epilepticus models and MTLE patients and may vary among protocols.

Kindling was used to stimulate the hippocampus, amygdala (McIntyre, Nathanson et al., 1982; Lehmann, Ebert et al., 1998; Brandt, Ebert et al., 2004), olfactory regions (Sutula, Cascino et al., 1989) or other areas of the brain (Cavazos and Sutula, 1990; Inoue, Morimoto et al., 1992) repeatedly over days to weeks to induce seizures in rats. Subsequent kindling treatments elicited progressively increasing electroencephalographic seizure after-discharge and behavioral seizures. Several months after kindling, animals responded with evoked seizures to subconvulsant stimulation. Among the chemoconvulsants, pentylenetetrazole (PTZ) a GABA receptor antagonist (Sutula, Cavazos et al., 1992) and the GABA_A ergic antagonist, bicuculline methiodide (Uemura and Kimura, 1988) have been used for kindling.

Electrical stimulation is more commonly used than chemoconvulsants to induce kindling and allows focused stimulation of key neuroanatomic sites via electrode placement. Cavazos et al., (1994) stereotaxically implanted stainless steel bipolar electrodes in the perforant path near the angular bundle, amygdala, or olfactory bulb of anesthetized rats. After 2 weeks’ recovery, unanesthetized rats were kindled with a 1-sec train of 62 Hz biphasic constant current 1.0 msec square wave pulses to produce stage 5 seizures. Once the rats demonstrated stage 5 evoked seizures, they were deemed fully kindled. The amygdala is by far the most responsive region to kindling. No differences were observed in generalized seizure threshold or after-discharge durations in two groups of rats kindled in cortical versus basolateral amygdala; however, rats stimulated in the cortical amygdala took longer to attain stage 5 seizures (Gilbert, Gillis et al., 1984).

Seizures induced by kindling have been classified into five stages based on clinical signs. This scale was later adopted to describe seizures in other animal models of epilepsy (Racine, 1972). However, in the kindling models, seizures are evoked and spontaneous motor seizures are rare (McNamara 1984). Thus, kindling models are not representative of MTLE seizures, but some of the induced lesions may be useful for study.

Amygdaloid kindling caused neuronal necrosis and loss with massive gliosis in the ipsilateral hemisphere extending from the medial olfactory bulb through the amygdala-piriform cortex to the ventral hippocampus, especially CA1 fields, and to midline thalamic nuclei (Inoue, Morimoto et al., 1992). Unilateral neuronal loss in ipsilateral CA3 and bilateral loss in CA1 were observed after status epilepticus induced by kindling-like electrical stimulation of the deep pre-piriform cortex in rats (Cavazos and Sutula, 1990; Cavazos, Das et al., 1994). Represa and Ben-Ari (1992) counted neurons utilizing quantitative stereological methods following kindling by perforant path stimulation. Neuronal loss increased in the dentate hilus with increasing numbers of kindled generalized tonic clonic seizures; however, the lesions bore only slight resemblance to those reported in MTLE.

Sutula et al., (1992) described aberrant MF sprouting in the inner molecular layer of the dentate gyrus with no other hippocampal damage following electrical kindling. Similar sprouting occurred after generalized tonic-clonic seizures in rats following systemic administration of pentylenetetrazole (Golarai et al., 1992).

The primary advantage of kindling is that specific areas of the brain can be stimulated in a controlled manner. This, and the lack of HS, makes kindling a useful model for investigating the mechanisms underlying paradoxical TLE or TLE without HS. The disadvantages of kindling as a model of MTLE are the cumbersome nature of stereotactic electrode implantation or chemical inoculation into the brain, the long time needed for induction, the lack of spontaneous seizures, and the dissimilarity of lesions compared with MTLE.

Tetanus Toxin

Compared to post-status epilepticus and kindling models, less information is available about tetanus toxin models, which suggests lesser acceptance of these models. Tetanus toxin blocks neurotransmitter release in the brain. Stereotactic inoculation of tetanus toxin into the brain has been used to reproduce some features of MTLE; however, the resultant seizures are either weak or temporary, and lesions differ from those in the post-status epilepticus models or human MTLE patients.

In naïve rats, following perforant path stimulation, dentate granule cells filter and block any seizure discharges from spreading to other parts of the temporal lobe. Tetanus toxin blocks GABA_B receptors, and diminishes the ability of the dentate granule cells to block the spread of seizure discharges (Finnerty, Whittington et al., 2001). The reported neurobehavioral alterations and histopathologic findings in this animal model of epilepsy vary widely. When tetanus toxin was injected into the hippocampus of rats, (Mellanby, Hawkins et al., 1984), the induced spontaneous motor seizures were reversible and without associated lesions, but the rats had memory and learning deficits.

Jefferys and Williams (1987) reported that the evoked responses from CA3 pyramidal cells were depressed for several weeks after tetanus toxin administration. Spatial reference memory tasks in these rats were also impaired. Tetanus toxin injection into the parietal neocortex of rats caused excessive synchronization of neuronal activity in parietal and temporal areas leading to epileptic activity (Brener, Amitai et al., 1991). Although the seizures produced by tetanus toxin were spontaneous motor seizures, the lack of a pronounced motor component made it difficult to score the epileptic activity (Jefferys, Borck et al., 1995).

In tetanus toxin models, the limited distribution and intensity of reported lesions correlated with the brief duration of seizures (Toth, Yan et al., 1998; Jiang, Duong et al., 1999; Bender, Dube et al., 2003). Lesions were often confined to areas within and immediately adjacent to the injection site. The extent of reported neuronal damage varied from none to as much as 30% of CA1 pyramidal neurons, although the latter represented an area near the injection site (Bagetta, Corasaniti et al., 1990; Bagetta, Corasaniti et al., 1991). When injected into the dentate gyrus, tetanus toxin-induced lesions were limited to a marked loss of granule cells in the ipsilateral dentate gyrus (Jefferys, Evans et al., 1992). The intense neurodegeneration reported in ipsilateral dentate gyrus was probably a direct effect of tetanus toxin, because dentate granule cells generally exhibited only subtle degenerative change in other models of MTLE.

The low intensity and transient nature of spontaneous motor seizures, along with the dissimilarity of lesions to those in MTLE, limits the usefulness of the tetanus toxin model. Nonetheless, this model may facilitate study of the role of GABA_B receptors in epileptogenesis (see Section 4).

Hyperthermia

Many adults with TLE had a childhood history of febrile convulsions (Vestergaard et al., 2007). Whether prolonged febrile seizures changed the excitability of the limbic system and caused subsequent TLE remains to be determined. It is proposed that the early febrile seizures damage the hippocampus leading to HS and consequent development of MTLE in adulthood (Harvey et al., 1995). Since brain development in rats 1–2 weeks of age is similar to that of human infants, hyperthermia is used to model MTLE.

Hyperthermia is especially useful for studying the culmination of febrile seizures in MTLE (Holtzman, Obana et al., 1981; Hjeresen, Guy et al., 1983; Hjeresen and Diaz, 1988; Baram, Gerth et al., 1997; Lemmens, Lubbers et al., 2005; Scantlebury, Gibbs et al., 2005). Ambient hyperthermia for six- and 10-day-old rat pups produced seizures that caused electrocortical paroxysmal discharges (Holtzman, Obana et al., 1981). Microwave energy (Hjeresen, Guy et al., 1983) also increased the core temperatures of 13- and 17-day-old rats to cause convulsions similar to febrile convulsions in human infants. A regulated stream of mildly heated air induced hyperthermia in 10- to 11-day-old rats (Baram, Gerth et al., 1997); about 93% of the rats developed stereotypical seizures. Although hyperthermic treatment caused immediate seizures, spontaneous motor seizures, as reported in post status epilepticus animal models and in human MTLE patients, did not occur in this model.

There are few reports of the neuropathologic changes of febrile seizure model rats. Rats were placed in a water-bath at 45°C for 4 minutes (Jiang, Duong et al., 1999) resulting in both visual and electroencephalographic seizures ranging in duration from 30 s to 6 min; seizure duration increased with the number of seizures. In most rats, no neurodegeneration was detected, although there was aberrant MF sprouting of granule cell collaterals into the inner molecular layer of the dentate gyrus. A few rats with evolution of short seizures into status epilepticus had neuronal degeneration in the hippocampus, temporal cortex, and mediodorsal thalamus. Findings were similar in another model in which hyperthermia was induced using a warmed air stream above the animals (Toth, Yan et al., 1998).

Neuronal argyrophilia, indicating physicochemical changes, and scattered apoptotic neurons were present in the central nucleus of the amygdala and CA1 and CA3 subfields of the hippocampus up to 2 weeks following seizures (Lowenstein, Thomas et al., 1992; Golarai, Greenwood et al., 2001; Santhakumar, Ratzliff et al., 2001; D’Ambrosio, Fender et al., 2005). Bender et al., (2004) performed serial MRIs at several time-points on immature rats exposed to hyperthermia and recorded altered T2 values in the dorsal hippocampus, the piriform cortex, and amygdala, without any evidence of neuronal degeneration, as assessed using the Fluoro-Jade histologic staining method. It was concluded that, although neuronal degeneration was not present, the affected areas may have neuronal alterations that produced seizures. The lesions have limited similarities to those reported in post-status epilepticus models and humans.

In contrast to the definitive spontaneous motor seizures in adult MTLE human patients, spontaneous limbic seizures do not occur during adulthood in the hyperthermic-seizure models (Dube, Chen et al., 2000), which casts doubt on their merit. Moreover, lesions in hyperthermic seizure models have limited resemblance to those in MTLE or post status epilepticus models. Therefore, the utility of this model relative to MTLE may be limited.

Post-Traumatic Epilepsy Model

Traumatic brain injury, with associated reduction in hippocampal volume (Bigler, Blatter et al., 1997; Tate and Bigler, 2000), is one of the most frequent causes of human TLE (Salazar, Jabbari et al., 1985; Annegers, Hauser et al., 1998). Trauma-associated MTLE has been partially replicated in post-traumatic epilepsy animal models. These models have produced spontaneous motor seizures as reported in MTLE. However, in contrast to the mesial temporal cortical origin of spontaneous motor seizures in MTLE and post-status epilepticus models, the seizures in post-traumatic epilepsy models originate in the frontal-parietal cortex and progress to the mesial temporal cortex.

In post-traumatic models of TLE, brain trauma has been produced by fluid percussion injury (FPI) (Lowenstein, Thomas et al., 1992; Golarai, Greenwood et al., 2001; D’Ambrosio, Fender et al., 2005) or cortical weight drop injury (Weisend and Feeney 1994; Golarai, Greenwood et al., 2001). Santhakumar et al., (2001) used electrophysiology in the rat FPI model to reveal that a low-frequency, single-shock stimulation of the perforant path led to early granule cell hyperexcitability with return to normal by 1 month. However, a persistent decrease in threshold to induction of seizure-like electrical activity was observed in response to high-frequency tetanic hippocampal stimulation in those rats. A brief (10 ms) pressure pulse of 3.75–4 atm was delivered directly to the intact dura through a 3-mm wide burr hole in the skull (D’Ambrosio, Fairbanks et al., 2004). Transient dural compression by a single episode of severe FPI resembled human cases of closed head injury and led to post-traumatic epilepsy. In this model, chronic electrocorticography revealed partial spontaneous motor seizures that originated from the neocortex at the injury site, deteriorated progressively, and spread to other parts of the neocortex. The same laboratory subsequently reported seizure origin from the frontal-parietal cortex that progressed to the mesial temporal cortex (D’Ambrosio, Fender et al., 2005). The seizures originated only from the mesial temporal neocortex in the post status epilepticus models of MTLE.

There are several descriptions of pathologic alterations in post-traumatic rat models of epilepsy (Chiba 1985; Jensen, Applegate et al., 1991; Jensen, Holmes et al., 1992; Lowenstein, Thomas et al., 1992; Jensen, Blume et al., 1995; Jensen, Wang et al., 1998; D’Ambrosio, Fender et al., 2005). The number of dentate hilar neurons was markedly decreased without abnormality of CA neurons 1 week post-trauma in a FPI model (Golarai, Greenwood et al., 2001). These lesions were confirmed by another study (Santhakumar, Ratzliff et al., 2001), which also described marked ipsilateral necrosis of pyramidal neurons in CA3 with milder necrosis in CA1 neurons and granule cells after weight drop contusion. Timm’s stain-positive MF sprouting was recorded in the dentate gyrus in the FPI model (D’Ambrosio, Fender et al., 2005). Two to four weeks post-FPI, remarkable neuronal loss and calcification were present in the ipsilateral thalamus; CA1 and CA3 hippocampal subfields exhibited loss of pyramidal neurons similar to that in human patients suffering from FPI (Chiba 1985; Jensen, Applegate et al., 1991; Jensen, Holmes et al., 1992; Jensen, Blume et al., 1995; Jensen, Wang et al., 1998). Gliosis was present in the ipsilateral hippocampus and temporal cortex.

Post-traumatic models have been useful in studying trauma-associated MTLE in humans. These protocols have produced spontaneous motor seizures as reported in MTLE; however, in contrast to the mesial temporal cortical origin of spontaneous motor seizures in MTLE and post-status epilepticus models, the seizures originated in the frontal-parietal cortex and progressed to the mesial temporal cortex in the post-traumatic epilepsy model (D’Ambrosio, Fender et al., 2005). The difference in seizure origin, the inconsistent development of hippocampal lesions that resemble those of post-status epilepticus models and MTLE, the high morbidity and mortality, and the labor-intensive nature of the procedures make post-traumatic models less desirable than post-status models for replicating MTLE.

Perinatal Hypoxia/ischemia

Perinatal HI has been associated with increased risk of epilepsy during early childhood or later (Watanabe, Kuroyanagi et al., 1982; Bergamasco, Benna et al., 1984). However, the relationship of perinatal HI to the development of seizures is poorly understood. Perinatal HI has been used in rats to investigate one of the initiating injuries causing epileptogenesis in MTLE. However, this treatment is inconsistent at producing spontaneous motor seizures or histopathologic lesions.

In a rat model of perinatal hypoxia, brief, moderate global hypoxia (3–4% O₂) at postnatal day (P) 10–12 led to spontaneous tonic clonic seizures. Hypoxia induced at younger or older ages did not lead to seizures, suggesting the age-dependent epileptogenic potential of hypoxia (Jensen, Applegate et al., 1991). Rat pups exposed to hypoxia during P10-12 exhibited minimal to no histopathologic lesions, but had increased susceptibility to convulsant-induced seizures during adulthood (Jensen, Applegate et al., 1991; Jensen, Holmes et al., 1992; Lowenstein, Thomas et al., 1992). Global hypoxia caused an acute increase in excitability in the immature brain and hypoxia during postnatal day 10 led to persistent hippocampal susceptibility to seizures. The age-dependent epileptogenic effects of hypoxia were partly due to a direct and permanent effect on neuronal excitability in the hippocampus (Jensen, Wang et al., 1998). The increased susceptibility of CA1 pyramidal neurons is likely related to the presence of greater numbers of glutamate receptors on these neurons (McDonald and Johnston 1990). These findings are corroborated by another observation that the non-N-methyl-D-aspartate (NMDA) antagonist 6-nitro-7-sulfamoylbenzo (f) quinoxaline-2,3-dione (NBQX) blocks the acute and chronic epileptogenic effects of global hypoxia in a perinatal HI model (Jensen, Blume et al. 1995).

Because hypoxia alone cannot induce spontaneous motor seizures, it has been combined with ischemia (Williams, Dou et al., 2004). In one study utilizing combined hypoxia/ischemia, approximately 50% of 7-day-old rats exhibited spontaneous motor seizures and 20% died without exhibiting any seizures, after they were kept in a chamber filled with 8% oxygen following ligature of the right common carotid artery.

Although there are several reports about electrophysiological and behavioral studies in the perinatal hypoxia/ischemia model of epilepsy (Lowenstein, Thomas et al., 1992; Towfighi and Mauger, 1998; Williams, Dou et al., 2004), the literature characterizing the morphological changes is minimal. The reported lesions, although similar to those of MTLE patients and post-status epilepticus models, are inconsistently produced (Towfighi and Mauger 1998). Neuronal necrosis was observed in the CA1 through CA4 regions of the hippocampus, lateral and medial nuclei of the dorsal thalamus, nucleus reticularis of the thalamus, striatum, medial habenular nucleus, and olfactory tubercle (Williams, Dou et al., 2004). These lesions, which were limited to the ipsilateral hippocampus following ligation of the right common carotid artery and subsequent hypoxemia, decreased with age and were no longer present in pups older than 13 days. Increased Timm’s staining (evidence for mossy fiber sprouting) was observed in the inner molecular layer of the dentate gyrus in about half of the 7-day-old rats that exhibited spontaneous motor seizures following a similar technique of hypoxia-ischemia induction (Liu, Mikati et al., 1995).

Opportunities to investigate the initiating epileptogenic injury in MTLE and the ease of induction are advantages of the perinatal HI model. However, high mortality, limited induction of spontaneous seizures and the inconsistent and less extensive MF sprouting as compared to the status epilepticus models and changes in human MTLE reduce the acceptability of this model.

Influence of Age/Strain Selection on Response to Epileptogenic Treatments

In rodents, both age and strain affected the response to epileptogenic treatments. Immature rats were more susceptible to SE than were adult rats. In immature (18-day-old) rats, KA and kindling caused relatively mild and no hippocampal neuronal degeneration, respectively. Neither treatment caused aberrant MF sprouting. In contrast, in mature rats, both KA and kindling caused milder seizures, but more severe hippocampal neuronal degeneration and aberrant MF sprouting (Haas et al., 2001). Despite apparent age differences, no strain differences in susceptibility to epileptogenic agents have been documented for rats. In a study in progress, the authors did observe more severe neurodegeneration in CA1 as compared to CA3 of F-344 rats than those previously reported in SD rats; however, differences may have been due to slight differences in KA exposure. In contrast, mice exhibited strain-based variations in susceptibility to epileptogenic treatments, as evidenced by development of hippocampal neuronal degeneration and aberrant MF sprouting in 129/SvEMS mice and lack of such lesions in ICR mice following KA administration (McNamara et al., 1996; Schauwecker and Steward 1997; Cantallops and Routtenberg 2000). The relatively severe hippocampal neuronal degeneration and altered synaptic physiology may sensitize adult rats and susceptible mouse strains to epileptogenic stimuli and development of spontaneous seizures.

Conclusions and future directions

We have reviewed the commonly used animal models, the clinicopathologic features, and the leading mechanistic hypotheses of MTLE. Current evidence suggests that the initial SE, rather than the subsequent repeated spontaneous seizures, causes many of the hippocampal lesions (Gorter et al., 2003). Although extensive evidence supports the central role of aberrant MF sprouting, its exact contribution to the development of MTLE has not yet been fully characterized. In our experience using KA in F-344 rats, aberrant MF sprouting precedes the onset of spontaneous motor seizures. In MTLE, aberrant MF sprouting may be an endpoint triggered by childhood injury followed by a latency period. The damaged hippocampus undergoes many additional changes including neuronal degeneration, dentate granule cell neurogenesis, microgliosis, astrocytic hyperplasia (astrocytosis) and hypertrophy (astrogliosis), and sprouting of axons of CA1 pyramidal neurons. The latency period is probably characterized by a complex interplay of multiple factors, such as immediate early genes, second messengers, transcription factors, protein translation, alterations in receptors and ion channels, axonal and dendritic plasticity, neuronal loss, neurogenesis and gliosis. Hence, we should consider the possibility that even if aberrant MF sprouting is the primary cause, it may not be the only cause of spontaneous motor seizures in MTLE.

This review focused on histopathologic endpoints. New imaging methods such as functional MRI, PET scanning, and single photon emission computed tomography will provide additional insight. Furthermore, immunohistochemistry and molecular methods will be critical to exploring the complex mechanisms of epileptogenesis in MTLE. Microarray gene profiling will undoubtedly generate prolific data, which will be more meaningful if arrays are coupled with in-situ hybridization or immunohistochemistry to localize the selected genes or their products at the tissue level. Individual mechanistic pathways can then be dissected by using RNA silencing (siRNA) techniques and/or knock-out mice models.

Animal models, such as the kainate and pilocarpine models of the post-status epilepticus class, display spontaneous motor seizures like those in human patients. In contrast, kindling models mainly exhibit evoked seizures. Some animal models, especially the post-status epilepticus models, develop lesions which appear to be identical to those in human MTLE. Based on the similarity of lesions (e.g., neuronal degeneration and loss in hippocampus, aberrant MF sprouting and gliosis), a latency period, and the subsequent development of spontaneous motor seizures, post-status epilepticus models have received the greater acceptance as MTLE models. However, these models also develop bilateral neuronal loss not only in the limbic areas including the hippocampus and amygdala, but also in extralimbic regions such as thalamus, hypothalamus, and certain areas of the cerebral cortex. The significance of these lesions in other parts of the brain needs further investigation.

Among post-status epilepticus models, systemic KA and pilocarpine models are relatively easy to produce, whereas electric stimulation models are more laborious and cumbersome. The kindling, tetanus toxin, hyperthermia, post-traumatic epilepsy and perinatal HI models exhibit some of the salient features of MTLE. However, kindling leads to evoked (rather than spontaneous) seizures and spontaneous motor seizures (as observed in MTLE) are poorly induced in tetanus toxin and perinatal HI models and transient in adult hyperthermia model rats.

Understanding the cellular and molecular mechanisms of epileptogenesis requires animal models that mimic the genesis of MTLE in human patients. Some important effects of spontaneous motor seizures such as memory loss and behavioral alterations are difficult to evaluate in current models of MTLE. Nonetheless, these animal models will remain valuable research tools because the presence of an intact central nervous system allows study of pathogenic mechanisms of MTLE and provides opportunities to evaluate potential antiepileptogenic drugs.